2,2-bis(hydroxymethyl)butanoic acid

Endpoint summary

Description of key information

Acute oral

Performed according to EU Method B.1 (Acute Toxicity (Oral)) guideline and in compliance with GLP,

Groups (5/sex/dose) of CD rats of Sprague-Dawley origin (Hsd:Sprague-Dawley(CD)) were given a single oral (gavage) dose of DMBA at 2000 mg/kg bw. Animals were then observed for mortality, clinical signs and bodyweights for 14 days.

No mortality was observed. Clinical signs of reaction to treatment comprised piloerection, hunched posture (in all animals), soft to liquid faeces (in all males and three females) and increased salivation (in one female). There were no other signs of reaction to treatment and recovery was complete in all rats by Day 8. All animals were considered to have achieved satisfactory bodyweight gains throughout the study. No abnormalities were observed at the study termination necropsy. In this study, the combined oral LD50 of DMBA was considered to be higher than 2000 mg/kg bw in rats.

Under the test conditions, the oral LD50 for DMBA is higher than 2000 mg/kg bw in rats therefore it is not classified according to the CLP Regulation (EC) N° (1272-2008).

 

Acute inhalation toxicity

The first acute inhalation toxicity study with 2,2-bis(hydroxymethyl)butanoic acid was carried out in accordance to the OECD Guideline 436: Acute inhalation toxicity – Acute toxic class method (2009) in GLP conditions.

The study was performed with 3 males and 3 females of rats (Rattus norvegicus, strain Wistar) for each tested group. Concentration of test material in air (vehicle) was: nominal concentration = 4.5 mg/L (Group 1) and 9.6 mg/L (Group 2) , achieved mean concentration (measured in breathing zone using gravimetric and analytical techniques was 1.11 mg/L or 24.7 % of output (Group 1) and 3.67 mg/L or 38.2 % of output (Group 2).
Type of inhalation exposure was Head-Nose only and the animals were adapted to the exposure tubes for 5 days with gradually increased time to a total duration of 4 hours by the fifth day.
All the rats were observed for mortality and clinical signs during exposure and daily during the subsequent 14-day post-exposure period
The maximal cut-off value of exposure concentration 5 mg/L was failed to reach achieving a respirable particle size (MMAD = 1-4 μm). For Group 2, exposure was performed with a feasible concentration of 3.67 ± 0.51 mg/L with a maximal analytical value of 4.6 mg/L for one sample.
There were no animal’s morbidity and mortality on 1.11 ± 0.25 mg/L and 3.67 ± 0.51 mg/L exposure. Clinical signs in some animals treated with lower and higher concentrations were observed 24-48 hours after exposure and considered to be caused by the irritative effect of the test item. The total weight gain in all animals was positive, except for one female in group 1, which showed a fall in body weight on the last day of the study for an unclear reason. No gross findings were revealed during necropsy.
Based on effects on the maximal feasible concentration 3.67 ± 0.51 mg/L that was lower than the cut-off value 5 mg/L, the acute inhalation LC50 in rats is considered to be more than 5 mg/L.

The second acute inhalation toxicity study with the test item (DMBA)  was carried out in accordance to the OECD Guideline 403: Acute inhalation toxicity (2009) in GLP conditions.

The concentration level was used in this limit test corresponds to the maximum feasible concentration achieved previously by generating dust aerosol from a pelletized substance under a 12 bar pressure scraped off in a dust generator at a speed of 0.1 mm/min and Flow application 15 L/min, Flow air 5 L/min. At the same conditions, but with the lower relative humidity in the inhalation chamber, the mean feasible concentration of exposure was 3.2 mg/L.
There were no animal’s morbidity and mortality and significant clinical signs on this concentration; the total weight gain in all animals was positive. The decrease in body temperature and respiration rate 24-48 hours after exposure was reversible. No gross findings were revealed during necropsy two weeks after exposure and no test item related microscopic findings were found in the nasal cavity, nasopharynx, paranasal sinus, larynx, trachea and lungs.
Based on effects on the maximal feasible concentration 3.2 ± 0.4 mg/L and 3.2 ± 0.3 mg/L (for males and females), the acute inhalation LC50 in rats is considered to be more than cut-off value 5 mg/L.

 

 

Acute dermal toxicity

 

The test of acute dermal toxicity to the rat (strain Wistar) for DMBA was performed as a limit test according to 92/69/EWG, B.3 (OECD 402) under GLP condition.  5 females and 5 males of rats were exposed with semiocclusive type of coverage to dose of 2000 mg/kg bw for 24 hours.

Results: No mortalities occurred and no clinical symptoms were observed. The body weight gain was within the range commonly recorded for animals of this strain and age.

No effects to the organs were observed. Only slight scaling of the treated skin was observed in one male and two females, which persisted in two animals (one female and one male) until day 11 and in one female animal until day 12 of the observation period.

The LD50 was established as more than 2000 mg/kg bw. (LD 50 > 2000 mg/kg bw).

Key value for chemical safety assessment

Acute toxicity: via oral route

Link to relevant study records

Reference

Endpoint:

acute toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1996-11-12 to 1996-11-26

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: GLP study conducted with minor deviations: temperature in the experimental room was outside the target range

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

EU Method B.1 (Acute Toxicity (Oral))

Deviations:

yes

Remarks:

temperature in the experimental room was outside the target range

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes

Test type:

standard acute method

Limit test:

yes

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan UK Ltd, Bicester, Oxon, England.
- Age at study initiation: Approximately 4-7 weeks
- Weight at study initiation: 75-89 g
- Fasting period during the study: Animals were fasted for overnight period before test item administration and for approximately 4 h after dosing.
- Housing: Animals were housed in groups of 5/sex in metal cages with wire mesh floors
- Diet: Standard laboratory rodent diet (SDS R&M 1), ad libitum
- Water: Drinking water, ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature: 17-19.5 °C
- Humidity: 39-59 %
- Air changes: 10-15 air changes/hour
- Photoperiod: 12 h dark / 12 h light

IN-LIFE DATES: From: 1996-11-12 To: 1996-11-26

Route of administration:

oral: gavage

Vehicle:

other: distilled water

Details on oral exposure:

VEHICLE
- Concentration in vehicle: 20 % w/v

MAXIMUM DOSE VOLUME APPLIED: 10 mL/kg bw

DOSAGE PREPARATION: DMBA was formulated on the day of dosing at a concentration of 20 % w/v in distilled water.

Doses:

2000 mg/kg bw

No. of animals per sex per dose:

5

Control animals:

no

Details on study design:

- Duration of observation period following administration: 14 days
- Frequency of observations: Cages of rats were checked at least twice daily for any mortalities. Animals were observed soon after dosing and at frequent intervals for the remainder of Day 1. On subsequent days animals were observed once in the morning and again at the end of the experimental day (with the exception of Day 15 - morning only).
- Frequency of weighing: Bodyweight of each rat was recorded on Days 1 (prior to dosing), 8 and 15.
- Necropsy of survivors performed: Yes; all animals were killed on Day 15 by cervical dislocation and were subjected to a macroscopic examination.

Statistics:

None

Sex:

male/female

Dose descriptor:

LD50

Effect level:

> 2 000 mg/kg bw

Based on:

test mat.

Remarks on result:

other: No mortality was observed.

Mortality:

No mortality was observed.

Clinical signs:

other: - Piloerection was observed in all rats within five minutes of dosing; later on Day 1 it was accompanied by hunched posture in all animals and soft to liquid faeces in all males and three females. In addition, one female showed increased salivation within

Gross pathology:

No abnormalities were observed at the study termination necropsy.

Other findings:

None

None

Interpretation of results:

other: not classified as dangerous for oral acute toxicity according to Regulation (EC) No 1272/2008 (CLP Regulation)

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

Under the test conditions, the oral LD50 for DMBA is higher than 2000 mg/kg bw in rats therefore it is not classified according to the Annex VI to the Directive 67/548/EEC and the CLP Regulation (EC) N° (1272-2008).

Executive summary:

Test Guidance

Performed according to EU Method B.1 (Acute Toxicity (Oral)) guideline and in compliance with GLP,

Method

Groups (5/sex/dose) of CD rats of Sprague-Dawley origin (Hsd:Sprague-Dawley(CD)) were given a single oral (gavage) dose of DMBA at 2000 mg/kg bw. Animals were then observed for mortality, clinical signs and bodyweights for 14 days.

Results

No mortality was observed. Clinical signs of reaction to treatment comprised piloerection, hunched posture (in all animals), soft to liquid faeces (in all males and three females) and increased salivation (in one female). There were no other signs of reaction to treatment and recovery was complete in all rats by Day 8. All animals were considered to have achieved satisfactory bodyweight gains throughout the study. No abnormalities were observed at the study termination necropsy. In this study, the combined oral LD50 of DMBA was considered to be higher than 2000 mg/kg bw in rats.

Conclusion

Under the test conditions, the oral LD50 for DMBA is higher than 2000 mg/kg bw in rats therefore it is not classified according to the CLP Regulation (EC) N° (1272-2008).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

LD50

Value:

> 2 000 mg/kg bw

Acute toxicity: via inhalation route

Link to relevant study records

Reference

Endpoint:

acute toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 436 (Acute Inhalation Toxicity: Acute Toxic Class Method)

Version / remarks:

Acute Toxic Class Method, 07 September, 2009.

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Certificate of GLP Compliance No G-044 from 17.07.2021, valid until 17.07.2023, Slovak National Accreditation Service

Test type:

acute toxic class method

Species:

rat

Strain:

Wistar

Remarks:

Wistar, outbreed

Sex:

male/female

Details on test animals or test system and environmental conditions:

6 males and 6 females

Source: The Lab Animals Breeding Center, Branch of the Institute of Bioorganic Chemistry RAS (www.spf-animals.ru) Nauki 6, Pushchino, Moscow region, Russia

Characteristic:
Nulliparous and non-pregnant animals with defined microbiological health status. The animal health monitoring is performed by breeder under FELASA-guidelines quarterly in AnLab, s.r.o. (Czech Republic). Health monitoring report from the breeder is attached to the study materials.

Date of batch receiving and age:
26.08.2021, 18 males of 5 weeks old, and 18 females of 7 weeks old

Age at day of exposure: Males: 7-8 weeks old; Females: 9-10 weeks old

Body weight at day of exposure:
Males: 222 ± 5 g, N = 6
Females: 199 ± 2 g, N = 6

Water: Tap water filtered with MilliRO system was provided ad libitum in standard water bottles.

Diet: Animals were fed Laboratory Rodent Diet ad libitum

Environmental Conditions
- actual mean temperature ranged from 21 °C to 24 °C
- mean relative humidity ranged from 30 % to 70 %.

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

nose/head only

Vehicle:

air

Mass median aerodynamic diameter (MMAD):

>= 1 - <= 4 µm

Remark on MMAD/GSD:

Group 1: 2.84 ± 3.06 μm (sample 1) and 3.03 ± 2.73 μm (sample 2)
Group 2: 3.57 ± 2.89 μm (sample 1) and 3.74 ± 2.98 μm (sample 2)

Details on inhalation exposure:

For the inhalation, rats (3/sex/level) were placed in exposure tubes connected via the ports to the inhalation chamber of the TSE Inhalation System.
Food and water were withdrawn from rats during exposure.

Each aerosol exposure consisted of a 30-minute T95 equilibration period, a 240-minute exposure period and a 30-minute de-equilibration period equal to the T95 equilibration period. T95 equilibration period was established in the preliminary sighting tests and constantly monitored by online data recording.
After each aerosol exposure, animals were removed from the exposure tubes and returned to the home cage with ad libitum feed and water.
In Step 1, the animals selected to Group No.1 (3 males and 3 females) were exposed at the 1.1 mg/L expected level.
In Step 2, exposure was performed with a maximum feasible concentration that has reached the actual level 4 mg/L and provided the respirable particle size (MMAD 1-4 μm).
The period of four days between the exposures at each concentration level has been allowed for the observation of delayed toxicity

Analytical verification of test atmosphere concentrations:

yes

Duration of exposure:

4 h

Remarks on duration:

Inhalation (Head-Nose only). The animals were adapted to the exposure tubes for 5 days with gradually increased time to a total duration of 4 hours by the fifth day.
Post-exposure period: 14 days

Concentrations:

Concentration of test material in vehicle:

Nominal concentration = 4.5 mg/L (Group 1) and 9.6 mg/L (Group 2). Achieved mean concentration was 1.11 mg/L or 24.7 % of output (Group 1) and 3.67 mg/L or 38.2 % of output (Group 2).

No. of animals per sex per dose:

3 males and 3 females (Group 1),
3 males and 3 females (Group 2).

Control animals:

other: A concurrent negative control group is not necessary according to OECD 436.

Statistics:

Body weight means and standard deviations were calculated separately for males and females.

Calculation of the mean particle size (mass median aerodynamic diameter, MMAD) with geometric standard deviation (GSD), and percentage of particles <4.0 μm were done using PSD (Particle Size Distribution for Windows) software, TSE System.

Test item should be allocated to one of four hazard categories based on acute toxicity estimate (ATE) according to the numeric cut-off criteria of the GHS for dusts and mists [2]:
Category 1: ATE < 0.05 mg/L
Category 2: 0.05 < ATE < 0.5 mg/L
Category 3: 0.5 < ATE < 1.0 mg/L
Category 4: 1.0 < ATE < 5.0 mg/L
The method of ATE – LC50 was not applied due to lack of mortality.

Key result

Sex:

male/female

Dose descriptor:

LC50

Effect level:

> 5 mg/L air

Based on:

test mat.

Exp. duration:

4 h

Remarks on result:

other: Based on effects on the maximal feasible concentration 3.67 ± 0.51 mg/L that was lower than the cut-off value 5 mg/L, the acute inhalation LC50 in rats is considered to be more than 5 mg/L.

Mortality:

There were no mortality in the Group 1 and Group 2 male and female rats exposed to a 4-hour inhalation to the test item 2,2-bis(hydroxymethyl)butanoic acid at concentrations 1.11 ± 0.25 mg/L and 3.67 ± 0.51 mg/L air.

Clinical signs:

other: In Group 1, the most animals showed ocular/nasal discharge on day 1 (after end of exposure until 3 hours). In Group 2, the ocular/nasal discharge was recorded in one female until day 2, one female had post-inhalation noisy breathing, and the pallor appear

Body weight:

The reduction in body weight gain was observed in two of three females in Group 1 and two of three females in Group 2. The mean total body weight gain was slightly lower in males inhaled with high concentration than in males treated with low concentration. However, the final mean body weight gain was positive in Groups 1 and 2.

Gross pathology:

During scheduled necropsy on day 15, no gross findings in respiratory organs (nasopharynx, trachea, lungs) and other organs were found.

Interpretation of results:

other: not classified as dangerous for inhalation acute toxicity according to Regulation (EC) No 1272/2008 (CLP Regulation)

Conclusions:

The inhalatory LC50 (4 hours) for the test item 2,2-bis(hydroxymethyl)butanoic acid in male and female rats is considered to be > 5 mg/L.

Executive summary:

The acute inhalation toxicity study with 2,2-bis(hydroxymethyl)butanoic acid was carried out in accordance to the OECD Guideline 436: Acute inhalation toxicity – Acute toxic class method (2009) in GLP conditions.

The study was performed with 3 males and 3 females of rats (Rattus norvegicus, strain Wistar) for each tested group. Concentration of test material in air (vehicle) was: nominal concentration = 4.5 mg/L (Group 1) and 9.6 mg/L (Group 2) , achieved mean concentration (measured in breathing zone using gravimetric and analytical techniques was 1.11 mg/L or 24.7 % of output (Group 1) and 3.67 mg/L or 38.2 % of output (Group 2).
Type of inhalation exposure was Head-Nose only and the animals were adapted to the exposure tubes for 5 days with gradually increased time to a total duration of 4 hours by the fifth day.
All the rats were observed for mortality and clinical signs during exposure and daily during the subsequent 14-day post-exposure period
The maximal cut-off value of exposure concentration 5 mg/L was failed to reach achieving a respirable particle size (MMAD = 1-4 μm). For Group 2, exposure was performed with a feasible concentration of 3.67 ± 0.51 mg/L with a maximal analytical value of 4.6 mg/L for one sample.
There were no animal’s morbidity and mortality on 1.11 ± 0.25 mg/L and 3.67 ± 0.51 mg/L exposure. Clinical signs in some animals treated with lower and higher concentrations were observed 24-48 hours after exposure and considered to be caused by the irritative effect of the test item. The total weight gain in all animals was positive, except for one female in group 1, which showed a fall in body weight on the last day of the study for an unclear reason. No gross findings were revealed during necropsy.
Based on effects on the maximal feasible concentration 3.67 ± 0.51 mg/L that was lower than the cut-off value 5 mg/L, the acute inhalation LC50 in rats is considered to be more than 5 mg/L.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

LC50

Value:

> 5 mg/L air

Physical form:

other: Dust aerosol

Acute toxicity: via dermal route

Link to relevant study records

Reference

Endpoint:

acute toxicity: dermal

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Qualifier:

according to guideline

Guideline:

OECD Guideline 402 (Acute Dermal Toxicity)

Qualifier:

according to guideline

Guideline:

other: 92/69/EWG, B.3

GLP compliance:

yes

Limit test:

yes

Species:

rat

Strain:

Wistar

Remarks:

(SPF)

Sex:

male/female

Type of coverage:

semiocclusive

Vehicle:

other: Bidestilliertes Wasser "ENGLISH" bidist. water

Duration of exposure:

24 hours

No. of animals per sex per dose:

5

Key result

Sex:

male/female

Dose descriptor:

LD50

Effect level:

> 2 000 mg/kg bw

Mortality:

Male: 2000 mg/kg bw; Number of animals: 5; Number of deaths: 0
Female: 2000 mg/kg bw; Number of animals: 5; Number of deaths: 0

Clinical signs:

other: no mortalities occurred. No clinical symptoms were observed.

Gross pathology:

Effects on organs:
No effects to the organs were observed.

Other findings:

Signs of toxicity (local):
Slight scaling of the treated skin was observed in one male and two females, which persisted in two animals (one female and one male) until day 11 and in one female animal until day 12 of the observation period.

Interpretation of results:

other: not classified as dangerous for dermal acute toxicity according to Regulation (EC) No 1272/2008 (CLP Regulation)

Conclusions:

No adverse effect was observed.

Executive summary:

The test of acute dermal toxicity to the rat (strain Wistar) for DMBA was performed as a limit test according to 92/69/EWG, B.3 (OECD 402) under GLP condition.  5 females and 5 males of rats were exposed with semiocclusive type of coverage to dose of 2000 mg/kg bw for 24 hours.

Results: No mortalities occurred and no clinical symptoms were observed. The body weight gain was within the range commonly recorded for animals of this strain and age.

No effects to the organs were observed. Only slight scaling of the treated skin was observed in one male and two females, which persisted in two animals (one female and one male) until day 11 and in one female animal until day 12 of the observation period.

The LD50 was established as more than 2000 mg/kg bw. (LD 50 > 2000 mg/kg bw).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

LD50

Value:

> 2 000 mg/kg bw

Justification for classification or non-classification

Under the test conditions, the oral LD50 for DMBA is higher than 2000 mg/kg bw in rats therefore it is not classified according to the CLP Regulation (EC) N° (1272-2008).

Under the test conditions, the dermal LD50 for DMBA is higher than 2000 mg/kg bw in rats therefore it is not classified according to the CLP Regulation (EC) N° (1272-2008).

Under the test conditions and based on effects on the maximal feasible concentration 3.2 ± 0.4 mg/L and 3.2 ± 0.3 mg/L (for males and females), the acute inhalation LC50 in rats is considered to be more than cut-off value 5 mg/L therefore it is not classified according to the CLP Regulation (EC) N° (1272-2008).